ABSTRACT
Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019(T), which are known to possess 7alpha-HSDH activity. The level of 7alpha-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7alpha-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.
Subject(s)
Bacteroides/metabolism , Chenodeoxycholic Acid/metabolism , Cholic Acid/metabolism , Feces/microbiology , Adult , Bacterial Typing Techniques , Bacteroides/classification , Bacteroides/growth & development , Bacteroides/isolation & purification , Bacteroides fragilis/metabolism , Biotransformation , Chromatography, Thin Layer , Culture Media/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Deoxycholic Acid/analogs & derivatives , Deoxycholic Acid/metabolism , Escherichia coli/metabolism , Female , Humans , Lithocholic Acid/analogs & derivatives , Lithocholic Acid/metabolism , Male , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Young AdultABSTRACT
In humans and animals, intestinal flora is indispensable for bile acid transformation. The goal of our study was to establish gnotobiotic mice with intestinal bacteria of human origin in order to examine the role of intestinal bacteria in the transformation of bile acids in vivo using the technique of gnotobiology. Eight strains of bile acid-deconjugating bacteria were isolated from ex-germ-free mice inoculated with a human fecal dilution of 10(-6), and five strains of 7alpha-dehydroxylating bacteria were isolated from the intestine of limited human flora mice inoculated only with clostridia. The results of biochemical tests and 16S rDNA sequence analysis showed that seven out of eight bile acid-deconjugating strains belong to a bacteroides cluster (Bacteroides vulgatus, B. distasonis, and B. uniformis), and one strain had high similarity with Bilophila wadsworthia. All five strains that converted cholic acid to deoxycholic acid had greatest similarity with Clostridium hylemonae. A combination of 10 isolated strains converted taurocholic acid into deoxycholic acid both in vitro and in the mouse intestine. These results indicate that the predominant bacteria, mainly Bacteroides, in human feces comprise one of the main bacterial groups for the deconjugation of bile acids, and clostridia may play an important role in 7aplha-dehydroxylation of free-form primary bile acids in the intestine although these strains are not predominant. The gnotobiotic mouse with bacteria of human origin could be a useful model in studies of bile acid metabolism by human intestinal bacteria in vivo.
